JP3453144B2 - Method for producing 1-deamino-8-D-arginine vasopressin - Google Patents

Abstract

A process for the manufacture of 1-deamino-8-D-arginine vasopressin (DDAVP) comprising, condensing a preparation of Mpa(R1)-Tyr-Phe-Gln-Asn-Cys(R2)-Pro-OH (SEQ ID NO: 1), where R1 and R2 are sulfhydryl-protecting groups, with the dipeptide (R3)-D-Arg(HCl)-Gly-NH2, where R3 is an acid-sensitive amino-protecting group, to form Mpa(R1)-Tyr-Phe-Gln-Asn-Cys(R2)-Pro-D-Arg(HCl)-Gly-NH2 (SEQ ID NO: 1), which is oxidized with iodine in a protic solvent. The reaction mixture containing the oxidized product can be purified by ion exchange chromatography on a cation exchange resin equilibrated with acid. Also disclosed is high-purity DDAVP obtained by this process and its use for treating diurea.

Description

The present invention relates to a novel method for producing highly pure 1-deamino-8-D-arginine vasopressin used for diuretic therapy. This method uses Mpa (R ¹ ) -Tyr-Phe
Condensed ^{-Gln-Asn-Cys (R 2} ) -Pro-OH (SEQ specific ID NO: 1) with ^{(R 3) -D-Arg (} HCl) -Gly-NH 2, from oxidizing the resulting peptide Become.

Hormonal analogue 1-deamino-8-D-arginine
Vasopressin (1-β-mercaptopropionic acid, 8
-D-arginine) -vasopressin or desmopressin, which will be abbreviated as "DDAVP" hereinafter, is an important antidiuretic agent for treating diuresis associated with diabetes insipidus, nocturnal enuresis, and urinary incontinence.

1-deamino-8-D-arginine vasopressin replaces the terminal cystine half with the 1-β-mercaptopropionic acid and replaces arginine with D-arginine, compared with vasopressin of the nervous pituitary in the living body. However, in this specification,
The name is “nonapeptide derivative”. Similarly, the starting materials and intermediates for the synthesis will be named n-peptide derivatives. Here, n is equal to the total number of amino acids and pseudo amino acid halves in each peptide.

Homogeneous phase (US Pat. No. 3,497,491 to Zaoral et al.),
Solid phase (Krchnak, V. and Zaoral M., 1979, Coll.Czechosl
ov. Chem. Commun. 44 : 1173-1178) has already been described. However, 1-deamino-8-D-arginine vasopressin is only obtained in amorphous form by conventional methods and is therefore difficult to purify. When used for therapeutic purposes, impurities, such as those with pressor effects, cause complications.

It was previously known in the art that yields increase at the expense of purity, and vice versa, and thus the larger the product molecule, the more costly the purification is. . In the pharmaceutical field, high purity is required as a premium, so that conventionally, a wide range of emphasis has been placed on purity. However, the conventional method requires an excessive purification step, so that the yield is inevitably reduced. Purification is costly in terms of manpower and time, and leads to a reduction in product yield, resulting in a clear loss of profit.

Therefore, there is a need in this field for DDAVP manufacturing synthetic routes that increase the yield of the final steps of the synthesis. This reduces the need for extensive purification steps for intermediates, products, improves yields and provides superior products. Furthermore, in this field, impurities that are relatively uncomplicated and have a pressure-increasing effect or other unfavorable biological effects are not formed so much or can be easily removed in the purification process.
There is a demand for improved methods of DDAVP manufacturing.

Therefore, it is an object of the present invention to provide a high-yield production method of highly pure 1-deamino-8-D-arginine vasopressin, that is, DDAVP.

Another object is to provide a simple, economical process for producing high yields of high purity DDAVP by avoiding complicated and expensive purification processes in the final step.

This invention comprises 1-deamino-8-D consisting of the following steps:
-A method for producing arginine vasopressin (I).
That is: a) heptapeptide Mpa (R ¹ ) -Tyr-Phe-Gln-Asn
Prepared -Cys (R2) -Pro-OH ( II) ( SEQ specific ID NO: 1), however, here, R ¹ and R ² are sulfhydryl-protecting groups are the same or different, b ) The above heptapeptide is replaced with dipeptide (R ³ ) -D-
Condensed with Arg (HCl) -Gly-NH ₂ (III), where R ³ is an acid-sensitive amino protecting group, c) nonapeptide Mpa (R ¹ ) -Tyr-Phe-Gln-Asn- C
ys (R ² ) -Pro-D-Arg (HCl) -Gly-NH ₂ (IV) (SEQ ID NO: 1) is formed, and d) This nonapeptide (IV) is a protic solvent or a solvent mixture. Iodide dissolved in.

A preferred method of preparing the heptapeptide (II) in step (a) is by condensing the tripeptide Mpa (R ¹ ) -Tyr-Phe-X (V). However, here, X is in place of the hydroxyl group in the carboxyl group of the Phe (-OH), the tetrapeptide ^{R 3 -Gln-Asn-Cys (} R 2) -Pro-OH (VI)
It is a reactive moiety substituted with.

The sulfhydryl protecting groups R ¹ and R ² may be the same, or independently of each other, acetamide methyl (Acm), tertiary-
Butyl, tertiary-butylsulfenyl, p-methylbenzyl, p-methoxybenzyl, 2- (3-nitropyridine-sulfenyl), ethylcarbamoyl, triphenylmethyl, or even 9-fluorenylmethyl Good.

According to a preferred embodiment of this invention, the sulfhydryl protecting groups R ¹ and R ² are both acetamidomethyl. Of the present invention, according to another preferred embodiment, the amino protecting group R ³ is N-3 grade - a butyloxy (Boc).

Further, the reactive moiety X is an activated ester of a carboxylic acid, such as an ester of p-nitrophenol or hydroxysuccinimide, or a non-activated ester of a carboxylic acid, such as a methyl, ethyl or benzyl ester. Alternatively, it is preferably azide. Thus, the reactive moiety X may be an alkoxy, aryloxy or azide, and the alkoxy is preferably succinimidoxy and the aryloxy is preferably n-nitrophenyloxy.

In the above method, the oxidation with I _{2 in} step (d) is carried out at pH 5.
It is desirable to carry out below 0, preferably between pH 2.5 and 4.3.

In this method, after all the synthesis is completed without interruption, the reaction mixture can be purified in almost the same state as obtained by the oxidation with iodine in step (d). A preferred purification route is via ion exchange chromatography on a cation exchange resin equilibrated with acid. Furthermore, the ion exchange resin used for purification preferably contains a sulfonic acid group. For purification, a gel chromatography step may be further added for finishing.

The present invention further discloses high-purity DDAVP prepared by the method of the present invention and its use in the treatment of diuresis caused by diseases such as diabetes insipidus, nocturnal enuresis and urinary incontinence.

The invention will now be described in more detail on the basis of preferred, but non-limiting embodiments. However, here
R ¹ and R ² are both acetamidomethyl, R ³ is N-tert-butyloxy, and X is —NHNH ₂ .

Example 1 Boc-Gln-Asn-Cys (Acm) -Pro-OH (X). BocCys
(Acm) ONp was prepared from BocCys (Acm) OH (Novabiochem, Leuferingen, CH) and p-nitrophenol by reaction with N, N'-dicyclohexylcarbodiimide (DCC) dissolved in ethyl acetate, Boc-Cys (Acm) -Pro
For the preparation of -OH (XII), this is reacted with H-Pro-OH dissolved in DMF / ethyl acetate / at 0 ° C and used without purification. During the reaction, Et3N is added to keep the pH neutral. yield
81%, purity> 95% (TLC).

Boc-Asn-Cys (Acm) -Pro-OH (XIII) was deblocked from XII in HCl / HOAC at room temperature and the resulting crude H-Cys (Acm) -Pro-OH.HCl was removed. Dissolve in DMF, neutralize it with Et ₃ N, and keep the pH at neutral while keeping the Boc at -5 ° C.
-Asn-ONp (Novabiochem, Leuferringen, CH) are added. XIII is isolated with a yield of 81%.
[Α] D ²⁰ = -77.1 ° (1 g per 100 ml DMF). The tetrapeptide derivative X was prepared by using XIII and Boc-Gln-ONp (Novabioche) in the same manner as in the case of preparing XIII from XII and Boc-Asn-ONp.
m, Leuferingen, CH). Yield 78.5
%, [Α] D ²⁰ = -89.7 ° (1 g per 100 ml H ₂ O).

Example 2 Mpa (Acm) -Tyr-Phe-NHNH2 (XI). Mpa (Acm) -Tyr
-OEt (XIV) was added to DMF containing 1-hydroxy-benzotriazole (HOBt) while keeping the pH at 7 (Et3
N) at 0 ° C., H-Tyr-OEt.HCl was added to Mpa (Acm) -OH (Bac
hem AG, CH) and DCC. Yield 48%. Hydrazide XI is a DMF / α-chymotrypsin enzyme.
In H ₂ O, it is prepared by the ester XIV is reacted with _{H-Phe-N 2 H 3} . Yield 90%. Melting point 240-242 [deg.] C.

Example 3 Mpa (Acm) -Tyr-Phe-Gln-Asn-Cys (Acm) -Pro-O
H (VII) (SEQ ID NO: 1) Boc-Gln-Asn-Cys (Ac
m) -Pro-OH (X) (11.7 g) is dissolved in 56 ml trifluoroacetic acid (TFA) and left at room temperature for 1 hour to evaporate. The residue is dissolved in 19 ml of dimethylformamide (DMF), cooled and the pH adjusted to 7 (Et3N). Mpa (Ac
m) -Tyr-Phe-NHNH ₂ (XI, 11.0 g) was dissolved in 115 ml DMF, the solution was cooled to -18 ° C and HCl / ethyl acetate (1
5.8 ml, 3.2M) is added and the solution is kept at -15 ° C. Isoamyl nitrite (3.5 ml) was added and the solution was heated to approximately -10 ° C.
Shake for 15 minutes. After cooling to −20 ° C., the pH is adjusted to 7 (Et ₃ N) The DMF solution of the tetrapeptide with the block removed is added and the mixture is shaken at −5 ° C. After the reaction was nearly complete (deblocked, quantified by TLC, tetrapeptide 99.5
% Conversion), the precipitated salts are filtered off and the volume of the solution is vacuum distilled to 60 ml. EtOH (99.5%, 21.5 ml) is added and the solution is heated to 60 ° C. After cooling to ambient temperature, pH
Is adjusted to 2.5 (HCl concentrate). The precipitate is filtered off and 9
After washing with 9.5% EtOH and drying, 17.6 g of white crystals are obtained. Yield 85%, melting point 188-191 ° C.

EXAMPLE 4 Boc-D-Arg (HCl ) -Gly-NH 2 (VIII). H-Gly-NH
2. Suspend HCl (2.6g) and 1-hydroxy-benzotriazole (HOBt, 3.3g) in 34ml DMF and cool the solution to 10 ° C. Et3N (2.0 ml) is added and the mixture is shaken for 15 minutes. Boc-D-arginine (HCl) (6.8 g) is added and the temperature is brought to 0 ° C. Add DCC (4.5 g) dissolved in 4.5 ml DMF and adjust the pH to 6.0 (Et3N). After complete conversion,
The precipitate formed is filtered off and the filtrate is distilled under reduced pressure.

Dissolve the residue in 80 ml of water, cool the solution to 0 ° C.,
To 3 (1M HCl). After filtering off the HOBt, the solution is extracted with dichloromethane. The aqueous phase is vacuum distilled to a volume of 20 ml and subjected to azeotropic distillation with butanol (4x),
The volume is brought to 60 ml by addition of BuOH. Solution, 10
0.1M HCl containing 5% NaCl (w / w) and 5% butanol (v / v)
, And then the volume is reduced to half by distillation.

After repeating the azeotropic distillation with butanol and filtering off the NaCl, the solution is poured into 8 volumes (v / v) of isopropyl acetate and the precipitate is collected by filtration and washed with isopropyl acetate. Compound VIII was obtained as a white amorphous powder. Yield 6.8 g (90%), [α] D ²⁰ = 4.3 °.

Example 5 Mpa (Acm) -Tyr-Phe-Gln-Asn-Cys (Acm) -Pro-
D-Arg (HCl) -Gly- NH 2 (IX) ( SEQ specific ID NO: 1).
BOC-D-Arg (HCl) -Gly-NH ₂ (VIII) (6.8g) 23m
Dissolve in l of acetic acid and add 20 ml HCl / HOAc (2.2M).
After shaking for 1.5 hours at room temperature, the solution is distilled under reduced pressure and the residue is dissolved in 30 ml DMF. The deblocked dipeptide is precipitated as an oily drop by adding xylene (31 ml). After decanting the supernatant, the residue is washed with xylene and the residual solvent is distilled under reduced pressure. The residue is dissolved in 110 ml DMF and the solution is cooled to -10 °. Adjust pH to 7.5 with Et3N.

Mpa (Acm) -Tyr-Phe-Gln-Asn-Cys (Acm) -Pro
-OH (VII) (SEQ ID NO: 1) (14g) and 2.1g HOBt
Dissolve in 65 ml DMF and DMF solution of the deblocking dipeptide HD-Arg (HCl) -Gly-NH ₂ obtained in the previous step,
Add the calculated amount of DCC. After 90% conversion (TLC), the DCU was filtered off, the solution was reduced to 100 ml volume and heated to 60 ° C, 3
Pour into 10 ml EtOH / EtOAc 85:15 (v / v).

The precipitate is collected by filtration and washed with EtOH / EtOAc 85:15. Compound IX was obtained as a white powder (yield 15.5 g (84.9 g
%), Melting point 182-185 ° C.) and purity was 94.5% (HPL
C).

Example 6 1-deamino-8-D-arginine vasopressin Mpn-Tyr-Phe-Gln-Asn-Cys-Pro-D-Arg-Gly-N
H ₂ (I) (acetate) (SEQ ID NO: 2). 10g Mpa (Ac
m) -Tyr-Phe-Gln-Asn-Cys (Acm) -Pro-D-Arg
(HCl) -Gly-NH ₂ (IX) (SEQ ID NO: 1) is dissolved in 10 l of acetic acid / water 1: 9 (v / v). 2 g of the blocked nonapeptide derivative IX dissolved in 80 ml of ethanol at room temperature
Oxidize is added by using a roller pump.
The addition rate is adjusted by a UV monitor in order to keep the amount of free iodine in the reaction solution low. The yellow color persists after the entire amount of IX has been consumed.

The reaction solution containing the product was equilibrated with an aqueous acetic acid solution at 1.61.
Pass through a short glass column containing S-Sepharose FF (Pharmacia, Sweden). 1-deamino-
8-D-arginine vasopressin (I) is 0.08M
Elute with NH ₄ Ac / AcOH buffer (241, pH 4.1, fractions monitored by HPLC analysis). Fractions containing DDAVP (I) in excess of 99% purity (ignoring those from the buffer) are pooled. The solution containing the pure title compound acetate is concentrated by reverse osmosis and finally lyophilized. White feathery powder.

DDAVP (I) is gel filtered on Sephadex G25 (0.1M acetic acid) for further purification. After elution, separate individual fractions by HPLC
Analyze by. Fractions containing pure product are pooled and lyophilized. > 99% pure DDAVP (I) is obtained in the form of the diacetate salt, with a yield of about 5 g. White feathery powder. yield
53%.

Example 7 DDAVP comparative test prepared according to Examples 1-6
And 1-deamino-8 prepared by the known homogeneous phase method
-D-arginine vasopressin.

The overall yield of DDAVP synthesized according to the homogeneous phase method disclosed in US Pat. No. 3,497,491 (starting from β-mercaptopropionic acid) is 5.0%.
The corresponding overall yield of 1-deamino-8-D-arginine vasopressin (I) (including the adjustment of Mpa (Acm) -OH) prepared by the process of the present invention was 7.0%.
It is an improvement of 0%. The method of the present invention having a synthetic route starting from β-mercaptopropynoic acid is (Boc-Cys
(Including adjustment of (Acm) -OH), overall yield 10.7%, 1-
Deamino-8-D-arginine vasopressin was produced.

More importantly, high yields were obtained in the last three steps of the process of the invention, namely 38% for compound X and 45% for compound VIII. . In known processes, the yields in this last three steps are usually in the range of about 8.5% -17% or about 10-20%. From an economic point of view of the synthetic method, the product loss in the final stage is more costly than in the initial stage, so the method is much more cost-effective.

According to the method of the present invention, the DD
Not only can AVP be produced in substantially high yield, but it can also be produced in substantially high purity.

In U.S. Pat. No. 3,497,491 there is no exact description of the purity of the final product. That is, the content of pure DDAVP in the final product is not given. Moreover, the presented results of basic analysis and amino acid analysis are only effective for confirmation of the structure. Further, although the optical rotation can be measured in principle strictly, the value of -65 ± 2 ° in [α] D ²⁵ described therein is not useful.

However, the information regarding pressor action in the third table of US Pat. No. 3,497,491 may be useful for assessing purity. 1-deamino-8-D-arginine vasopressin prepared according to US Pat. No. 3,497,491 is described as having considerable pressor side effects. Subsequent studies have revealed that 1-deamino-8-D-arginine (I) does not have such a pressor effect when strictly purified. However, further purification can only be done at the expense of yield, as long as the methods known in the art are followed.

The pressor side effects caused by impurities in products prepared by known methods include pharmacological complications such as vascular hypertension. Therefore, "purity" is related to two aspects. First, in the final product,
The actual content of active DDAVP, and secondly the content of medically harmful impurities. 1-deamino-8-D-arginine vasopressin (I) prepared by this method is almost free of such harmful impurities.

Although the invention has been fully described here,
Those familiar with this field will be able to modify the various steps of this process, the reagents used, and various reaction conditions to achieve similar results. Such modifications are within the scope and spirit of this disclosure.

Sequence list (i) General information (ii) Number of sequences: 2 Information on sequence identification number 1 (i) Sequence characteristics (A) Length: 4 Amino acids (B) Type: Amino acid (C) Configuration: Linear (ii) Molecular species : Peptide (iii) Sequence description: Sequence identification number 1 Information on sequence identification number 2 (i) Sequence characteristics (A) Length: 6 amino acids (B) Type: Amino acid (C) Configuration: Linear (ii) Molecular species: Peptide (iii) Sequence description: Sequence identification number 2

─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Melunstam, Eva Maria Birgitta Sweden country S-230 44 Bunkefro Strand Biernstopinna Legatán 35 (72) Inventor Rossie Star, Jan Anders in Gemal Sweden S-224-76 Runt Huikinga Huegen 21 Tue (72) Inventor Skert Betsk, Jan Aketh Sweden S-215 22 Malmo Abedei Sgatan 16 (56) Reference US Patent 3497491 (US, A) J Org Chem, 1978, 43 ( 25), 4800-4803 (58) Fields investigated (Int.Cl. ⁷ , DB name) C07K 7/16 C07K 1/107 A61K 38/11

Claims (9)

(57) [Claims] 1. A 1-deamino-8-D-comprising the following steps:
Synthesis of arginine vasopressin: a) the R ^1, R ² and sulfhydryl protecting groups, ^{heptapeptide, Mpa (R 1) -Tyr-} Phe-Gln-Asn-Cys (R 2) -Pr
o-OH (SEQ ID NO: 1) was added to tripeptide Mpa (R ¹ )-
Prepared by method consisting in condensing the Tyr-Phe-X, however, here, X is the location of the OH in -COOH of Phe, tetrapeptide ^{R 3 -Gln-Asn-Cys (} R 2) - Pro-OH
A reactive moiety substituted with b) the heptapeptide from step (a) is reacted with N, N′-dicyclohexyl-carbodiimide to give the dipeptide (R ³ ) -D-Arg (HCl) -Gly-NH 2. Condensed with ₂ , where R ³ is an acid sensitive amino protecting group, c) Nonapeptide Mpa (R ¹ ) -Tyr-Phe-Gln-Asn-Cys
(R ² ) -Pro-D-Arg (HCl) -Gly-NH ₂ (SEQ ID NO: 1) is formed, and d) the nonapeptide from step (c) is added to a pH below 5.0.
Then, it is oxidized by iodine in a protic solvent. 2. The sulfhydryl protecting groups R ¹ and R ² may be the same or, independently of each other, acetamidomethyl, tert-butyl, tert-butylsulfenyl, p
-Methylbenzyl, p-methoxybenzyl, 2- (3-
2. The method of claim 1, which may be selected from the group consisting of nitropyridine / sulfenyl), ethylcarbamoyl, triphenylmethyl, and 9-fluorenylmethyl. 3. The method according to claim 2, wherein the sulfhydryl protecting groups R ¹ and R ² are both acetamidomethyl. 4. The method according to claim 1, wherein the amino protecting group R ³ is N-tertiary-butyroxy. 5. The method of claim 1, wherein the reactive moiety is selected from the group consisting of alkoxy, aryloxy, and azide. 6. The method according to claim 5, wherein the alkoxy is succinimidoxy. 7. The method according to claim 5, wherein the aryloxy is p-nitrophenyloxy. 8. The process according to claim 1, wherein the oxidation with iodine is carried out between pH 1.5 and 4.3. 9. The process according to claim 1, wherein the reaction mixture containing the oxidized nonapeptide from step (d) is purified by ion exchange chromatography on a cation exchange resin equilibrated with acid.